The present invention is related to determining whether a human patient is susceptible to hereditary pancreatitis. More specifically, the present invention is related to determining whether a human patient is susceptible to hereditary pancreatitis by identifying a single G to A transition mutation in the third exon of cationic trypsinogen, or digesting the trypsinogen gene in exon III with Afl III.
Hereditary pancreatitis (HP) is an autosomal dominant disorder with 80% penetrance and variable expressivity [Perrault, J. Hereditary pancreatitis. Gastroenterol. Clin. North Am. 23:743-752 (1994); Madraso-de la Garza, J., Hill, I., Lebenthal, E. Hereditary pancreatitis. In: Go V, ed. The Pancreas: Biology, Pathobiology, and Disease. 2nd ed. New York: Raven, 1095-1101 (1993); Whitcomb, D. C., Preston, R. A., Aston, C. E., Sossenheimer, M. J., Barua, P. S., Zhang, Y., Wong-Chong, A., White, G., Wood, P., Gates, L. K., Jr., Ulrich, C., Martin, S. P., Post, J. C., and Ehrlich, G. D. A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology 110, 1975-1980 (1996); Bodic, L. L., Bignon, J. D., Raguenes, O., Mercier, B., Georgelin, T., Schnee, M., Soulard, F., Gagne, K., Bonneville, F., Muller, J. Y., Bachner, L., and Ferec, C. The hereditary pancreatitis gene maps to long arm of chromosome 7. Hum. Molec. Genet. 5, 549-554 (1996)]. Nearly 100 kindreds have been reported world-wide since the genetic nature of this disorder was recognized by Comfort and Steinberg in 1952 [Madraso-de la Garza, J., Hill, I., Lebenthal, E. Hereditary pancreatitis. In: Go V, ed. The Pancreas: Biology, Pathobiology, and Disease. 2nd ed. New York: Raven, 1095-1101 (1993); Comfort, M. and Steinberg, A. Pedigree of a family with hereditary chronic relapsing pancreatitis. Gastroenterology 21, 54 (1952)]. The majority of the families are of white European ancestry, but affected kindreds have been reported in Japan, India, and among other ethnic groups [Perrault, J. Hereditary pancreatitis. Gastroenterol. Clin. North Am. 23:743-752 (1994)]. HP is characterized by recurrent bouts of severe epigastric pain with onset, usually developing before ten years of age. The clinical, laboratory and pathologic features of HP are indistinguishable from attacks of pancreatitis from other causes. In addition to recurrent acute attacks, many HP patients progress to complicated chronic pancreatitis characterized by pancreatic calcifications, pseudocysts, chronic abdominal pain, pancreatic exocrine failure, diabetes mellitus and/or pancreatic cancer [Perrault, J. Hereditary pancreatitis. Gastroenterol. Clin. North Am. 23:743-752 (1994); Madraso-de la Garza, J., Hill, I., Lebenthal, E. Hereditary pancreatitis. In: Go V, ed. The Pancreas: Biology, Pathobiology, and Disease. 2nd ed. New York: Raven, 1095-1101 (1993)]. Despite years of research, no unique morphologic or biochemical markers have been identified for HP, and the pathophysiologic mechanisms that lead to intermittent attacks of acute pancreatitis remain obscure. Therefore, no rational or effective preventative strategies have been developed, and treatment consists solely of supportive care.
Because of the absence of biochemical markers specific for HP, attention has focused on identifying the HP disease gene. The availability of a high-density map of the human genome, based on polymorphic simple tandem repeat (STR) markers, and familial S0 linkage analysis made it possible to identify an HP gene locus within the q35 region of chromosome seven [Whitcomb, D. C., Preston, R. A., Aston, C. E., Sossenheimer, M. J., Barua, P. S., Zhang, Y., Wong-Chong, A., White, G., Wood, P., Gates, L. K., Jr., Ulrich, C., Martin, S. P., Post, J. C., and Ehrlich, G. D. A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology 110, 1975-1980 (1996); Bodic, L. L., Bignon, J. D., Raguenes, O., Mercier, B., Georgelin, T., Schnee, M., Soulard, F., Gagne, K., Bonneville, F., Muller, J. Y., Bachner, L., and Ferec, C. The hereditary pancreatitis gene maps to long arm of chromosome 7. Hum. Molec. Genet. 5, 549-554 (1996)]. It was thus desired to identify and sequence the HP gene to determine the site of the disease-causing mutation(s) in an effort to understand the molecular mechanism leading to HP. Several previously mapped genes on chromosome 7q were considered candidates for the HP disease gene because they are known to be expressed in the exocrine pancreas and encode enzymes that could potentially activate digestive enzymes within the pancreas. The hypothesis that pancreatitis results from inappropriate activation of pancreatic proenzymes was first promulgated 100 years ago and subsequently was demonstrated to be an experimental model for pancreatitis [Chiara, H. Ueber selbstverdauung des menschlichen pankreas. Zeitschrift fur heilkunde 17, 69-96 (1896); Steer, M. L., and Meldolesi, J. The cell biology of experimental pancreatitis. N. Engl. J. Med. 316 (3), 144-50, (1987)]. Although carboxypeptidase A1 (CPA1) was considered the primary candidate by Le Bodic [Bodic, L. L., Bignon, J. D., Raguenes, O., Mercier, B., Georgelin, T., Schnee, M., Soulard, F., Gagne, K., Bonneville, F., Muller, J. Y., Bachner, L., and Ferec, C. The hereditary pancreatitis gene maps to long arm of chromosome 7. Hum. Molec. Genet. 5, 549-554 (1996)], this gene mapped centromeric to the HP locus defined by obligate recombinations in an HP linkage study [Whitcomb, D. C., Preston, R. A., Aston, C. E., Sossenheimer, M. J., Barua, P. S., Zhang, Y., Wong-Chong, A., White, G., Wood, P., Gates, L. K., Jr., Ulrich, C., Martin, S. P., Post, J. C., and Ehrlich, G. D. A gene for hereditary pancreatitis maps to chromosome 7q35. Gastroenterology 110, 1975-1980 (1996); Stewart, E. A., Craik, C. S., Hake, L., and Bowcock, A. M. Human carboxypeptidase A identifies a Bg1II RFLP and maps to 7q31-qter. Am. J. Hum. Genet. 46 (4): 795-800, (1990); Rommens, J. M., Zengerling, S., Burns, J., Melmer, G., Kerem, B. S., Plavsic, N., Zsiga, M., Kennedy, D., Markiewicz, D., Rozmahel, R., et al. Identification and regional localization of DNA markers on chromosome 7 for the cloning of the cystic fibrosis gene. Am. J. Hum. Genet. 43 (5), 645-63 (1988); Rommens, J. M., Iannuzzi, M. C., Kerem, B., Drumm, M. L., Melmer, G., Dean, M., Rozmahel, R., Cole, J. L., Kennedy D., Hidaka, N., et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science. 245 (4922): 1059-65 (1989); Martise, T. C., Perlin, M., and Chakravarti, A. Automated construction of genetic linkage maps using an expert system (MultiMap): a human genome linkage map. Nature Genetics. 6 (4), 384-90 (1994)] and was, therefore, excluded from further consideration. However, at least eight trypsinogen genes are located on chromosome 7q35 between the STR markers D7S495 and D7S498 and within the V and D-C segments of the complex T-cell receptor xcex2 chain gene locus (TCRxcex2) [Rowen, L., Koop, B. F., Hood, L. The Complete 685-Kilobase DNA Sequence of the Human_T Cell Receptor Locus. Science (1996)]. Trypsinogen is an inactive proenzyme for trypsin, which becomes active when an eight amino acid amino-terminal peptide is removed. Although small amounts of trypsin are normally generated within the pancreas, this active trypsin is usually rapidly inactivated before pancreatic autodigestion occurs. Thus, the trypsinogen genes were considered primary candidates for the HP disease gene.
The entire 685 kilobase (kb) TCRxcex2-trypsinogen locus has recently been sequenced by Rowen et al., as part of the largest human genome sequencing project completed to date [Rowen, L., Koop, B. F., Hood, L. The Complete 685-Kilobase DNA Sequence of the Human_T Cell Receptor Locus. Science (1996) ]. As a result of this study, eight trypsinogen-like genes were sequenced and identified that map within the TCR xcex2 locus. Three were located at the 5xe2x80x2 end of the locus and were determined by sequence analysis to be pseudogenes. Another group of five trypsinogen genes, including the previously identified cationic and anionic pancreatic trypsinogen genes [Emi, M., Nakamura, Y., Owaga, M., et al. Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. Gene 41, 305-310 (1986); Tani, T., Kawashima, I., Mita, K., Takiguchi, Y. Nucleotide sequence of the human pancreatic tryspinogen III cDNA. Nucleic Acids Res 18, 1631 (1990); Weingand, U., Corbach, S., Minn, A., Kang, J., Muller-Hill, B. Cloning of the cDNA encoding human brain trypsinogen and characterization of its product. Gene 136, 167-175 (1993)], were found to be in a cluster located between the Vxcex24S1 and the Dxcex21 elements near the 3xe2x80x2 end of the TCRxcex2 locus. Based on comparisons with the pancreatic cDNAs in Genbank, one of the newly identified trypsinogen-like genes (T6 or TRYC, in the nomenclature of Rowen et al., reference 12) may also be functional, as it does not contain any stop codons or frameshift mutations within the predicted exonic regions. However, no corresponding cDNAs have been identified to date. The two other trypsinogen-like genes located within this 70 kilobase cluster are not likely to be expressed due to nonsense mutations (e.g. stop codons) and/or frameshift mutations [Rowen, L., Koop, B. F., Hood, L. The Complete 685-Kilobase DNA Sequence of the Human_T Cell Receptor Locus. Science (1996)].
These five trypsinogen genes are highly homologous, each residing within a tandemly duplicated 10 kb segment and each being composed of five exons. The extremely high degree of DNA sequence homology ( greater than 91%) present among this cluster of five trypsinogen genes demanded that highly specific sequence analysis strategies be developed for mutational screening. This was necessary to ensure that each sequencing run contained only the two alleles corresponding to a single gene, thereby permitting detection of heterozygotes, and not a dozen or more alleles from multiple related trypsinogen-like genes which would make detection of heterozygotes nearly impossible. The initial DNA sequencing effort focused on the trypsinogen genes that were known to be expressed, specifically cationic trypsinogen and anionic trypsinogen [Emi, M., Nakamura, Y., Owaga, M., et al. Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens. Gene 41, 305-310 (1986); Tani, T., Kawashima, I., Mita, K., Takiguchi, Y. Nucleotide sequence of the human pancreatic tryspinogen III cDNA. Nucleic Acids Res 18, 1631 (1990)] using members of the S-family [McElroy, R., and Christiansen, P. A. Hereditary pancreatitis in a kinship association with portal vein thrombosis. Am. J. Med. 52, 228-241 (1972)]. This strategy was accomplished by sequencing each of the five exons from the specifically cationic trypsinogen and anionic trypsinogen genes individually using a gene-specific, nested PCR strategy. Generation of mixed sequence data, by the simultaneous amplification of more than one of the closely related trypsinogen genes, was avoided by basing each of the two nested PCR primer sets used for each exon-specific amplification on minor differences within the introns. This provided for the direct amplification and sequencing of each exon from both the cationic and anionic trypsinogen genes, in their entirety, from genomic DNA. Comparison of the DNA sequences generated from the PCR amplifications of control specimens with the published sequence from these regions revealed 100% concordance, thus confirming the utility of the nested primer approach for gene-specific mutational screening.
The present invention pertains to a method for determining whether a human patient is susceptible to hereditary pancreatitis. The method comprises the steps of obtaining nucleic acid from the human patient. Then there is the step of checking the nucleic acid for a mutation that indicates hereditary pancreatitis.
The present invention pertains to a set of PCR primers which reacts with a human trypsinogen gene to identify hereditary pancreatitis. The primer is preferably either U306 GGTCCTGGGTCTCATACCTT (5xe2x80x2 outer) (SEQ ID NO 11), L1197 GGGTAGGAGGCTTCACACTT (3xe2x80x2 outer) (SEQ ID NO 15), U329 TGACCCACATCCCTCTGCTG (5xe2x80x2 inner/sequencing) (SEQ ID NO 12), or L924 TCTCCATTTGTCCTGTCTCT (3xe2x80x2 inner/sequencing) (SEQ ID NO 16) PCR amplification and cycle sequencing. Alternatively, the primers are TGTGAGGACATTCCTTGCGA (SEQ ID NO 5), TCTTCCTGAAAATTTTGACT (SEQ ID NO 18), ACAGAGACTTGGGAGCCACAGG (SEQ ID NO 6), GATACTTGCCTGCTTTTCTCA (SEQ ID NO 7), CGCCACCCCTAACATGCTATTG (SEQ ID NO 9), CCATCTTACCCAACCTCAGTAG (SEQ ID NO 10).
The present invention pertains to a method for detecting in a human a mutation in a trypsinogen gene indicative of hereditary pancreatitis. The invention comprises the steps of obtaining a sample having DNA of the patient. Then there is the 5 step of processing the sample so the trypsinogen genes within the DNA will be amplified by the specific primers. Next there is the step of introducing the desired restriction enzyme to the DNA wherein the digestion of the DNA by the desired restriction enzyme or direct DNA sequencing of the amplified gene indicates the presence of the mutation.
A G:A transition in the cationic trypsinogen gene is specifically associated with the HP phenotype. This mutation encodes an Arg:His substitution at residue 117, was observed in all HP affected individuals and obligate carriers from five kindreds; it was not observed in individuals who married into the families, nor was it observed in 140 unrelated individuals. X-ray crystal structure analysis, molecular modeling, and protein digest data suggest that the Arg 117 residue is a trypsin-sensitive site. Cleavage at this site is part of a fail-safe mechanism by which trypsin, that is activated within the pancreas, may be inactivated and that loss of this cleavage site would permit autodigestion resulting in pancreatitis.